This present invention relates to methods and kits for use in accelerating the proliferation and differentiation of hematopoietic and mesenchymal cells.
Bone marrow contains pluripotent stem cells that are capable of reconstituting either the hematopoietic system or a wide range of mesenchymal tissues. The mechanisms by which hematopoietic and mesenchymal stem cells produce a range of lineage-specific cell types are quite dissimilar.
a. The Hematopoietic System
The hematopoietic system is composed of a multitude of cell generations ranging from the terminally differentiated to very primitive hematopoietic lineage-specific cells, including a multipotent, self-renewing hematopoietic stem cell with long-term repopulating capability (HPC). (Traycoff, et al., Experimental Hematology 24:299-306, 1996). HPC are pluripotent lineage-specific cells that possess the ability to terminally differentiate into hematopoietic lineage-specific cells (HLSC). Hematopoiesis is an ongoing process, and therefore HPC must provide a continuous source of HLSC, which in turn can differentiate into red cells, platelets, monocytes, granulocytes and lymphocytes. (Prockop, Science 276:71-74, 1997). HPC proliferate either by xe2x80x9cself-renewalxe2x80x9d, to produce HPC-type progeny cells, or with accompanying differentiation, to produce HLSC. (Traycoff, et al., supra).
HPC transplantation therapy has been successful for a variety of malignant and inherited diseases and also provides myelopoietic support for patients undergoing high-dose chemotherapy or radiotherapy. (Emerson, Blood 87:3082-3088, 1996). However, stem cell transplantation has been limited by several features. First, acquiring a sufficient quantity of stem cells to achieve benefit after transfusion requires either extensive, operative bone marrow harvests or extensive pheresis procedures. (Emerson, supra). Next, even under these circumstances, only a limited number of useful cells is obtained. Finally, mature blood cell regeneration after transfusion is slow, so that little direct therapeutic benefit is seen for periods of 1 to 3 weeks. (Emerson, supra).
The development of in vitro culture techniques for hematopoietic cells combined with technologies for isolating relatively pure populations of HPC and HLSC has made possible their ex vivo expansion. (Alcorn and Holyoake, Blood Reviews 10:167-176, 1996, which is incorporated by reference herein). Successful ex vivo expansion of HPC, both by self-renewal and proliferation with differentiation, promises many clinical benefits, such as reduction of the number and duration of leucapheresis procedures required for autologous transplantation, thus reducing the risk of disease contamination in the apheresis products. (Alcorn and Holyoake, supra). Furthermore, ex vivo expansion may render inadequate HPC populations in peripheral blood and umbilical cord blood sufficient for autologous transplantation and adult allogeneic transplantation respectively. Finally, ex vivo expansion of HPC will greatly increase their utility as gene therapy vehicles. (Alcorn and Holyoake, supra). Similarly, ex vivo expansion of HLSC promises substantial clinical benefits, such as re-infusion of expanded populations of myeloid precursor cells to reduce the period of obligate neutropenia following autologous transplantation, the generation of natural killer cells for use in adoptive immunotherapy protocols, generation of megakaryocyte precursors to alleviate post-transplant-associated thrombocytopenia and more efficient generation of delivery systems for gene therapy. (Alcorn and Holyoake, supra).
Human bone marrow, umbilical cord blood, and peripheral blood lineage-specific cells mobilized by chemotherapy and/or cytokine treatment have been shown to be effective sources of HPC for transplantation following the administration of high-dose therapy to treat malignancy. (Holyoake, et al., Blood 87:4589-4595, 1996). Whatever the source of hematopoietic cells, most studies have used cultured cell populations selected on the basis of HPC-specific surface antigens, such as CD34. These cells can be readily obtained by a number of techniques. (Alcorn and Holyoake, supra). The results of several clinical trials using ex vivo expanded hematopoietic cells suggests that a fairly small number of HPC cultured ex vivo under appropriate conditions can initiate hematologic reconstitution. (Emerson, supra).
Survival and proliferation of HPC in ex vivo culture requires a combination of synergizing growth factors; the choice of cytokine/growth factor combination and culture system used will largely determine the fate of cells used to initiate the culture. (Alcorn and Holyoake, supra). In vivo, blood cell production is thought to be regulated locally by interactions of hematopoietic stem cells with a variety of cell-bound and secreted factors produced by adjacent bone marrow stromal cells. (Alcorn and Holyoake, supra). The addition of growth factors and cytokines to the culture medium is intended to compensate for the absence of stroma-associated activities. Growth factors and cytokines that have been shown to increase production of HPC (in various combinations) include granulocyte colony-stimulating factor (G-CSF), granulocyte/macrophage colony stimulating factor (GM-CSF), stem cell factor (SCF), macrophage colony-stimulating factor (M-CSF), and interleukins 1, 3, 6, and 11 (Reviewed in Takaku, J. Cancer Res. Clin. Oncol. 121:701-709, 1995; Holyoake, et al., supra). Conversely, inclusion of macrophage inhibitory protein-1xcex1 (MIP-1xcex1), tumor necrosis factor a (TNF-xcex1) or transforming growth factor xcex2 (TGFxcex2) in most expansion cultures reported to date results in decreased HPC and HLSC yields. (Emerson, supra).
A great deal of effort has gone into defining the optimal conditions for ex vivo culture of hematopoietic cells. Improved methods that increase of ex vivo proliferation rate of HPC will greatly increase the clinical benefits of HPC transplantation. This is true both for increased xe2x80x9cself-renewalxe2x80x9d, which will provide a larger supply of HPC capable of reconstituting the entire hematopoietic system, and for proliferation with differentiation, which will provide a larger supply of lineage-specific cells. Similarly, methods that increase in vivo proliferation of HPC will enhance the utility of HPC transplantation therapy by rapidly increasing local concentrations of HPC (and HLSC) in the bone marrow. Furthermore, methods that result in the differentiation of HPC and HLSC are useful in providing populations of specific cell types for use in cell therapy.
Transfection of mammalian HPC has been accomplished. (Larochelle, et al., Nature Medicine 2:1329-1337, 1996). Thus, methods that increase the proliferation of HPC and HLSC are also useful in rapidly providing a large population of transfected cells for use in gene therapy.
b. Mesenchymal Stem Cells
Mesenchymal stem cells (MSC) are pluripotent progenitor cells that possess the ability to differentiate into a variety of mesenchymal tissue, including bone, cartilage, tendon, muscle, marrow stroma, fat and dermis as demonstrated in a number of organisms, including humans (Bruder, et al., J. Cellul. Biochem. 56:283-294 (1994). The formation of mesenchymal tissues is known as the mesengenic process, which continues throughout life, but proceeds much more slowly in the adult than in the embryo (Caplan, Clinics in Plastic Surgery 21:429-435 (1994). The mesengenic process in the adult is a repair process but involves the same cellular events that occur during embryonic development (Reviewed in Caplan, 1994, supra). During repair processes, chemoattraction brings MSC to the site of repair where they proliferate into a mass of cells that spans the break. These cells then undergo commitment and enter into a specific lineage pathway (differentiation), where they remain capable of proliferating. Eventually, the cells in the different pathways terminally differentiate (and are no longer able to proliferate) and combine to form the appropriate skeletal tissue, in a process controlled by the local concentration of tissue-specific cytokines and growth factors (Caplan, 1994, supra).
Recently, it has been hypothesized that the limiting factor for MSC-based repair processes is the lack of adequate numbers of responsive MSC at the repair site (Caplan, 1994, supra). Thus, it has been suggested that by supplying a sufficient number of MSC to a specific tissue site the repair process can be controlled, since the repair site will supply the appropriate exposure to lineage-specific growth factors and differentiation molecules (Caplan, 1994, supra). Towards this end, several animal studies have demonstrated the feasibility of using autologous MSC for repair of various defects associated with mesenchymal tissue. (For review, see Caplan, et al., in The Anterior Cruciate Ligament: Current and Future Concepts, ed. D. W. Jackson, Raven Press, Ltd. NY pp.405-417 (1993). Recent work has demonstrated the feasibility of collection, ex vivo expansion in culture, and intravenous infusion of MSC in humans (Lazarus, et al., Bone Marrow Transplantation 16:557-564 (1995); Caplan and Haynesworth, U.S. Pat. No. 5,486,359, hereby incorporated by reference in its entirety). Further, MSC of animal origin have been transfected with retroviruses and have achieved high level gene expression both in vitro and in vivo (Allay, et al., Blood 82:477A (1993). Thus, the manipulation of MSC via such techniques seems a promising tool for reconstructive therapies and may be useful for gene therapy.
MSC therapy can serve as a means to deliver high densities of repair-competent cells to a defect site when adequate numbers of MSC and MSC lineage-specific cells are not present in vivo, especially in older and/or diseased patients. In order to efficiently deliver high densities of MSC to a defect site, methods for rapidly producing large numbers of MSC are necessary. MSC have been exposed to a number of growth factors in vitro, but only platelet-derived growth factor (PDGF) showed mitotic activity (Caplan et al., 1994, supra). Methods that increase the ex vivo proliferation rate of MSC will greatly increase the utility of MSC therapy. Similarly, methods that increase in vivo proliferation rate of MSC will enhance the utility of MSC therapy by rapidly increasing local concentrations of MSC at the repair site.
Furthermore, methods that enhance the proliferation rate of lineage-specific descendants of MSC, including but not limited to bone marrow stromal cells, osteoclasts, chondrocytes, and adipocytes, will enhance the therapeutic utility of MSC therapy by increasing the concentration of lineage-specific cell types at appropriate repair sites.
In one aspect, the present invention provides methods that promote hematopoietic stem and lineage-specific cell proliferation and differentiation, and mesenchymal stem and lineage-specific cell proliferation by contacting the cells with angiotensinogen, angiotensin I (AI), AI analogues, AI fragments and analogues thereof, angiotensin II (AII), AII analogues, AII fragments or analogues thereof or AII AT2 type 2 receptor agonists, either alone or in combination with other growth factors and cytokines.
In another aspect of the present invention, an improved cell culture medium is provided for the proliferation and differentiation of hematopoietic stem and lineage-specific cells, and for the proliferation of mesenchymal stem and lineage-specific cells, wherein the improvement comprises addition to the cell culture medium of an effective amount of angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII, AII analogues, AII fragments or analogues thereof or AII AT2 type 2 receptor agonists.
In a further aspect, the present invention provides kits for the propagation of hematopoietic and mesenchymal stem and lineage-specific cells, wherein the kits comprise cell an effective amount of angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII, AII analogues, AII fragments or analogues thereof or AII AT2 type 2 receptor agonists, and instructions for using the amount effective of active agent as a cell culture medium supplement. In a preferred embodiment, the kit further comprises a cell culture media. In another preferred embodiment, the kit further comprises a sterile container for cell culturing.
As defined herein, the term xe2x80x9cHPCxe2x80x9d refers to any hematopoietic pluripotent progenitor cells capable of giving rise to a wide variety of differentiated hematopoietic cell types. Cell types within this definition include, but are not limited to CD34+ bone marrow mononuclear cells (BMMC) (Berardi, et al., Blood 86:2123-2129, 1995), PBSC (Fritsch, et al., Bone Marrow Transplantation 17:169-178, 1996), cobblestone area forming cells (CAFC) (Lemieux, et al., Blood 86:1339-1347, 1995) and 5-FU BM cells (Alcorn and Holyoake, Blood Reviews 10:167-176, 1996). As defined herein, the term xe2x80x9cHLSCxe2x80x9d refers to hematopoietic lineage-specific cells, and includes the progeny of HPC that are committed to a cell-specific differentiation pathway, as well as fully differentiated hematopoietic cells. As defined herein, mesenchymal stem cells (MSC) are pluripotent progenitor cells that possess the ability to differentiate into a variety of mesenchymal tissue, including bone, cartilage, tendon, muscle, marrow stroma, fat and dermis, and include, but are not limited to, cells such as those described in Caplan and Haynesworth, U.S. Pat. No. 5,486,359. As defined herein, xe2x80x9cproliferationxe2x80x9d encompasses both cell self renewal and cellular proliferation with accompanying differentiation. As defined herein xe2x80x9cdifferentiationxe2x80x9d includes both entry into a specific lineage pathway and functional activation of differentiated cells.
U.S. Pat. No. 5,015,629 to DiZerega (the entire disclosure of which is hereby incorporated by reference) describes a method for increasing the rate of healing of wound tissue, comprising the application to such tissue of angiotensin II (AII) in an amount which is sufficient for said increase. The application of AII to wound tissue significantly increases the rate of wound healing, leading to a more rapid re-epithelialization and tissue repair. The term AII refers to an octapeptide present in humans and other species having the sequence Asp-Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:1]. The biological formation of angiotensin is initiated by the action of renin on the plasma substrate angiotensinogen (Clouston et al., Genomics 2:240-248 (1988); Kageyama et al, Biochemistry 23:3603-3609; Ohkubo et al., Proc. Natl. Acad. Sci. 80:2196-2200 (1983); each reference hereby incorporated in its entirety). The substance so formed is a decapeptide called angiotensin I (AI) which is converted to AII by the converting enzyme angiotensinase which removes the C-terminal His-Leu residues from AI [SEQ ID NO: 38]. AII is a known pressor agent and is commercially available.
Studies have shown that AII increases mitogenesis and chemotaxis in cultured cells that are involved in wound repair, and also increases their release of growth factors and extracellular matrices (diZerega, U.S. Pat. No. 5,015,629; Dzau et. al., J. Mol. Cell. Cardiol. 21:S7 (Supp III) 1989; Berk et. al., Hypertension 13:305-14 (1989); Kawahara, et al., BBRC 150:52-9 (1988); Naftilan, et al., J. Clin. Invest. 83:1419-23 (1989); Taubman et al., J. Biol. Chem. 264:526-530 (1989); Nakahara, et al., BBRC 184:811-8 (1992); Stouffer and Owens, Circ. Res. 70:820 (1992); Wolf, et al., Am. J. Pathol. 140:95-107 (1992); Bell and Madri, Am. J. Pathol. 137:7-12 (1990). In addition, AII was shown to be angiogenic in rabbit corneal eye and chick chorioallantoic membrane models (Fernandez, et al., J. Lab. Clin. Med. 105:141 (1985); LeNoble, et al., Eur. J. Pharmacol. 195:305-6 (1991). Therefore, AII may accelerate wound repair through increased neovascularization, growth factor release, reepithelialization and/or production of extracellular matrix. However, it is not known in the literature as to whether angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII, AII analogues and fragments thereof, or AII receptor agonists would be useful in accelerating the proliferation and differentiation of hematopoietic and mesenchymal stem and lineage-specific cells.
A peptide agonist selective for the AT2 receptor (AII has 100 times higher affinity for AT2 than AT1) has been identified. This peptide is p-aminophenylalanine6-AII [xe2x80x9c(p-NH2-Phe)6-AII)xe2x80x9d], Asp-Arg-Val-Tyr-Ile-Xaa-Pro-Phe [SEQ ID NO.36] wherein Xaa is p-NH2-Phe (Speth and Kim, BBRC 169:997-1006 (1990). This peptide gave binding characteristics comparable to AT2 antagonists in the experimental models tested (Catalioto, et al., Eur. J. Pharmacol. 256:93-97 (1994); Bryson, et al., Eur. J. Pharmacol. 225:119-127 (1992).
The effects of AII receptor and AII receptor antagonists have been examined in two experimental models of vascular injury and repair which suggest that both AII receptor subtypes (AT1 and AT2) play a role in wound healing (Janiak et al., Hypertension 20:737-45 (1992); Prescott, et al., Am. J. Pathol. 139:1291-1296 (1991); Kauffman, et al., Life Sci. 49:223-228 (1991); Viswanathan, et al., Peptides 13:783-786 (1992); Kimura, et al., BBRC 187:1083-1090 (1992).
Many studies have focused upon AII(1-7) (AII residues 1-7) or other fragments of AII to evaluate their activity. AII(1-7) elicits some, but not the full range of effects elicited by AII. Pfeilschifter, et al., Eur. J. Pharmacol. 225:57-62 (1992); Jaiswal, et al., Hypertension 19(Supp. II):II-49-II-55 (1992); Edwards and Stack, J. Pharmacol. Exper. Ther. 266:506-510 (1993); Jaiswal, et al., J. Pharmacol. Exper. Ther. 265:664-673 (1991); Jaiswal, et al., Hypertension 17:1115-1120 (1991); Portsi, et a., Br. J. Pharmacol. 111:652-654 (1994).
As hereinafter defined, a preferred class of AT2 agonists for use in accordance with the present invention comprises AII, AII analogues or active fragments thereof having p-NH-Phe in a position corresponding to a position 6 of AII. In addition to peptide agents, various nonpeptidic agents (e.g., peptidomimetics) having the requisite AT2 agonist activity are further contemplated for use in accordance with the present invention.
The active AII analogues, fragments of AII and analogues thereof of particular interest in accordance with the present invention comprise a sequence consisting of at least three contiguous amino acids of groups R1-R8 in the sequence of general formula I
xe2x80x83R1xe2x80x94R2xe2x80x94R3xe2x80x94R4xe2x80x94R5xe2x80x94R6xe2x80x94R7xe2x88x92R8
in which R1 and R2 together form a group of formula
Xxe2x80x94RAxe2x80x94RBxe2x80x94,
wherein X is H or a one to three peptide group and a peptide bond between RA and RB is labile to aminopeptidase A cleavage;
R3 is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc and Tyr;
R4 is selected from the group consisting of Tyr, Tyr(PO3)2, Thr, Ser, homoSer and azaTyr;
R5 is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly;
R6 is His, Arg or 6-NH2-Phe;
R7 is Pro or Ala; and
R8 is selected from the group consisting of Phe, Phe(Br), Ile and Tyr, excluding sequences including R4 as a terminal Tyr group.
Compounds falling within the category of AT2 agonists useful in the practice of the invention include the AII analogues set forth above subject to the restriction that R6 is p-NH2-Phe.
In one class of preferred embodiments, RA is suitably selected from Asp, Glu, Asn, Acpc (1-aminocyclopentane carboxylic acid), Ala, Me2Gly, Pro, Bet, Glu(NH2), Gly, Asp(NH2) and Suc. RB is suitably selected from Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-Lys. Particularly preferred combinations for RA and RB are Asp-Arg, Asp-Lys, Glu-Arg and Glu-Lys.
Particularly preferred embodiments of this class include the following: AII, AIII or AII(2-8), Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:2]; AII(3-8), also known as des1-AIII or AIV, Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:3]; AII(1-7), Asp-Arg-Val-Tyr-Ile-His-Pro {SEQ ID NO:4]; AII(2-7). Arg-Val-Tyr-Ile-His-Pro [SEQ ID NO:5]; AII(3-7), Val-Tyr-Ile-His-Pro [SEQ ID NO:6]; AII(5-8), Ile-His-Pro-Phe [SEQ ID NO:7]; AII(1-6), Asp-Arg-Val-Tyr-Ile-His [SEQ ID NO:8]; AII(1-5), Asp-Arg-Val-Tyr-Ile [SEQ ID NO:9]; AII(1-4), Asp-Arg-Val-Tyr [SEQ ID NO:10]; and AII(1-3), Asp-Arg-Val [SEQ ID NO:11]. Other preferred embodiments include: Arg-norLeu-Tyr-Ile-His-Pro-Phe [SEQ ID NO:12] and Arg-Val-Tyr-norLeu-His-Pro-Phe [SEQ ID NO:13]. Still another preferred embodiment encompassed within the scope of the invention is a peptide having the sequence Asp-Arg-Pro-Tyr-Ile-His-Pro-Phe [SEQ ID NO:31]. AII(6-8), His-Pro-Phe [SEQ ID NO:14] and AII(4-8), Tyr-Ile-His-Pro-Phe [SEQ ID NO:15] were also tested and found not to be effective.
Another class of compounds of particular interest in accordance with the present invention are those of the general formula II
R2xe2x80x94R3xe2x80x94R4xe2x80x94R5xe2x80x94R6xe2x80x94R7xe2x80x94R8
in which R2 is selected from the group consisting of H, Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-Lys;
R3 is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc and Tyr;
R4 is selected from the group consisting of Tyr, Tyr(PO3)2, Thr, Ser, homoSer and azaTyr;
R5 is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly;
R6 is His, Arg or 6-NH2-Phe;
R7 is Pro or Ala; and
R8 is selected from the group consisting of Phe, Phe(Br), Ile and Tyr.
A particularly preferred subclass of the compounds of general formula II has the formula
R2xe2x80x94R3-Tyr-R5-His-Pro-Phe [SEQ ID NO:16]
wherein R2, R3 and R5 are as previously defined. Particularly preferred is angiotensin III of the formula Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:2]. Other preferred compounds include peptides having the structures Arg-Val-Tyr-Gly-His-Pro-Phe [SEQ ID NO:17] and Arg-Val-Tyr-Ala-His-Pro-Phe [SEQ ID NO:18]. The fragment AII(4-8) was ineffective in repeated tests; this is believed to be due to the exposed tyrosine on the N-terminus.
In the above formulas, the standard three-letter abbreviations for amino acid residues are employed. In the absence of an indication to the contrary, the L-form of the amino acid is intended. Other residues are abbreviated as follows:
It has been suggested that AII and its analogues adopt either a gamma or a beta turn (Regoli, et al., Pharmacological Reviews 26:69 (1974). In general, it is believed that neutral side chains in position R3, R5 and R7 may be involved in maintaining the appropriate distance between active groups in positions R4, R6 and R8 primarily responsible for binding to receptors and/or intrinsic activity. Hydrophobic side chains in positions R3, R5 and R8 may also play an important role in the whole conformation of the peptide and/or contribute to the formation of a hypothetical hydrophobic pocket.
Appropriate side chains on the amino acid in position R2 may contribute to affinity of the compounds for target receptors and/or play an important role in the conformation of the peptide. For this reason, Arg and Lys are particularly preferred as R2.
For purposes of the present invention, it is believed that R3 may be involved in the formation of linear or nonlinear hydrogen bonds with R5 (in the gamma turn model) or R6 (in the beta turn model). R3 would also participate in the first turn in a beta antiparallel structure (which has also been proposed as a possible structure). In contrast to other positions in general formula I, it appears that beta and gamma branching are equally effective in this position. Moreover, a single hydrogen bond may be sufficient to maintain a relatively stable conformation. Accordingly, R3 may suitably be selected from Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc and Tyr.
With respect to R4, conformational analyses have suggested that the side chain in this position (as well as in R3 and R5) contribute to a hydrophobic cluster believed to be essential for occupation and stimulation of receptors. Thus, R4 is preferably selected from Tyr, Thr, Tyr (PO3)2, homoSer, Ser and azaTyr. In this position, Tyr is particularly preferred as it may form a hydrogen bond with the receptor site capable of accepting a hydrogen from the phenolic hydroxyl (Regoli, et al. (1974), supra).
In position R5, an amino acid with a xcex2 aliphatic or alicyclic chain is particularly desirable. Therefore, while Gly is suitable in position R5, it is preferred that the amino acid in this position be selected from Ile, Ala, Leu, norLeu, Gly and Val.
In the angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII analogues, fragments and analogues of fragments of particular interest in accordance with the present invention, R6 is His, Arg or 6-NH2-Phe. The unique properties of the imidazole ring of histidine (e.g., ionization at physiological pH, ability to act as proton donor or acceptor, aromatic character) are believed to contribute to its particular utility as R6. For example, conformational models suggest that His may participate in hydrogen bond formation (in the beta model) or in the second turn of the antiparallel structure by influencing the orientation of R7. Similarly, it is presently considered that R7 should be Pro in order to provide the most desirable orientation of R8. In position R8, both a hydrophobic ring and an anionic carboxyl terminal appear to be particularly useful in binding of the analogues of interest to receptors; therefore, Tyr and especially Phe are preferred for purposes of the present invention.
Analogues of particular interest include the following:
The polypeptides of the instant invention may be synthesized by any conventional method, including, but not limited to, those set forth in J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co., Rockford, Ill. (1984) and J. Meienhofer, Hormonal Proteins and Peptides, Vol. 2, Academic Press, New York, (1973) for solid phase synthesis and E. Schroder and K. Lubke, The Peptides, Vol. 1, Academic Press, New York, (1965) for solution synthesis. The disclosures of the foregoing treatises are incorporated by reference herein.
In general, these methods involve the sequential addition of protected amino acids to a growing peptide chain (U.S. Pat. No. 5,693,616, herein incorporated by reference in its entirety). Normally, either the amino or carboxyl group of the first amino acid and any reactive side chain group are protected. This protected amino acid is then either attached to an inert solid support, or utilized in solution, and the next amino acid in the sequence, also suitably protected, is added under conditions amenable to formation of the amide linkage. After all the desired amino acids have been linked in the proper sequence, protecting groups and any solid support are removed to afford the crude polypeptide. The polypeptide is desalted and purified, preferably chromatographically, to yield the final product.
Preferably, peptides are synthesized according to standard solid-phase methodologies, such as may be performed on an Applied Biosystems Model 430A peptide synthesizer (Applied Biosystems, Foster City, Calif.), according to manufacturer""s instructions. Other methods of synthesizing peptides or peptidomimetics, either by solid phase methodologies or in liquid phase, are well known to those skilled in the art.
Although AII has been shown to increase the proliferation of a number of cell types in vitro, it does not necessarily increase the proliferation of all cell types. Studies have shown that AII accelerates cellular proliferation through the production of transforming growth factor xcex2 (TGFxcex2) (Gibbons et al., J. Clin. Invest. 90:456-461 (1992). Thus, since only PDGF is known to be mitogenic for MSC, an ability of AII to effect MSC proliferation would be unexpected. Furthermore, as Emerson (supra) has shown that inclusion of TGF-xcex2 in most expansion cultures resulted in a decreased HPC and HLSC yield, it is unexpected that AII, through the action of TGF-xcex2, would be of benefit in such situations. No studies have reported that AII has an effect on the differentiation of either HPC or MSC.
In one aspect of the present invention, a method of increasing in vitro and ex vivo HPC and HLSC proliferation by exposure to angiotensinogen, angiotensin I (AI), AI analogues, AI fragments and analogues thereof, angiotensin II (AII), AII analogues, AII fragments or analogues thereof, or AII AT2 type 2 receptor agonists is disclosed. Experimental conditions for the isolation, purification, ex vivo growth and in vivo mobilization of HPC and HLSC have been reported (Berardi, et al., Blood 86(6):2123-2129, 1995; Fritsch, et al., Bone Marrow Transplantation 17:169-178, 1996; LaRochelle, et al., Nature Medicine 12:1329-1337, 1996; Traycoff, et al., Experimental Hematology 24:299-306, 1996; Holyoake, et al., Blood 87:4589-4595, 1996; Lemieux, et al., Blood 86:1339-1347, 1995; Talmadge, et al., Bone Marrow Transplantation 17:101-109, 1996; Bodine, et al., Blood 88:89-97, 1996; all references hereby incorporated by reference herein.)
In one embodiment of the invention, HPC are isolated from bone marrow, peripheral blood or umbilical cord blood. HPC is then selected for in these samples. HPC-enriched samples are cultured under appropriate growth conditions, in the presence of angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII, AII analogues, AII fragments and analogues thereof and/or AII AT2 type 2 receptor agonists. HPC proliferation is assessed at various time points during culture.
In a preferred embodiment, HPC and HLSC are isolated from bone marrow aspirates from the posterior iliac crest (Caplan and Haynesworth, U.S. Pat. No. 5,486,359). CD34+ HPC are isolated from the aspirate by attaching a biotinylated monoclonal antibody specific for CD34 (available from Becton Dickinson, Sunnyvale, Calif., USA) to a streptavidin affinity column (Ceprate SC; CellPro, Bothell, Wash., USA) and passing the aspirate through the column, followed by appropriate column washing and stripping, according to standard techniques in the art. CD34+ HPC are suspended in culture medium and incubated in the presence of, preferably, between about 0.1 ng/ml and about 1 mg/ml angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII, AII analogues, AII fragments and analogues thereof and/or AII AT2 type 2 receptor agonists. The cells are expanded for a period of between 8 and 21 days and cellular proliferation with accompanying differentiation is assessed via phase microscopy following standard methylcellulose colony formation assays (Berardi, et al., supra) at various points during this time period. Similarly, xe2x80x9cself-renewalxe2x80x9d of HPC is assessed periodically by reactivity to an antibody directed against CD34+.
In a further preferred embodiment, HPC that have been cultured in the presence of angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII, AII analogues, AII fragments and analogues thereof and/or AII AT2 type 2 receptor agonists are used for autologous transplantation, to reconstitute a depleted hematopoietic system. Prior to transplantation, the cells are rinsed to remove all traces of culture fluid, resuspended in an appropriate medium and then pelleted and rinsed several times. After the final rinse, the cells are resuspended at between 0.7xc3x97106 and 50xc3x97106 cells per ml in an appropriate medium and reinfused into a subject through intravenous infusions. Following transplantation, subject peripheral blood samples are evaluated for increases in the number of HPC, HLSC, and more mature blood cells at various time points by standard flow cytometry and cell sorting techniques. (Talmadge, et al., supra).
In another aspect of the present invention, a method of increasing in vitro and ex vivo MSC and lineage-specific mesenchymal cell proliferation by exposure to angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII, AII analogues, AII fragments and analogues thereof and/or AII AT2 type 2 receptor agonists is disclosed. Experimental conditions for the isolation, purification and in vitro growth of lineage-specific mesenchymal cells, such as bone-marrow derived stromal cells, have been reported (Johnson and Dorshkind, Blood 68(6):1348-1354 (1986); hereby incorporated by reference in its entirety). Other reports describe different conditions for culturing lineage-specific mesenchymal cells in vitro (Bab, et al., J. Cell Sci. 84:139-151 (1986); Benayahu, et al., J. Cellular Physiology 140:1-7 (1989); both references hereby incorporated by reference in their entirety).
In one embodiment of the present invention, MSC are isolated from bone marrow aspirates from the posterior iliac crest and/or femoral head cancellous bone, purified, resuspended in appropriate growth medium, counted and diluted to an appropriate concentration to seed in tissue culture plates (Caplan and Haynesworth, U.S. Pat. No. 5,486,359). Purified MSC are cultured in an appropriate growth medium and growth conditions in a humidified atmosphere. The cells are allowed sufficient time to attach to the tissue culture dish, whereupon non-attached cells are discarded. Adherent cells are placed in growth medium at 37xc2x0 C. in a humidified atmosphere in the presence of, preferably, between about 0.1 ng/ml and about 1 mg/ml angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII, AII analogues, AII fragments and analogues thereof and/or AII AT2 type 2 receptor agonists. The cells are expanded for a period of between 2 and 21 days and cellular proliferation is assessed at various time points during this time period. Subsequent medium changes are performed as needed. When the primary cultures are nearly confluent, the cells are harvested for reinfusion into a subject. Cells are examined microscopically to verify the absence of contamination. The cells are rinsed to remove all traces of culture fluid, resuspended in an appropriate medium and then pelleted and rinsed several times. After the final rinse, the cells are resuspended at between 0.7xc3x97106 and 50xc3x97106 cells per ml in an appropriate medium and reinfused into a subject through intravenous infusions. Subjects are evaluated for MSC proliferation in vivo by means of a repeat diagnostic bone marrow aspirate and biopsy to be compared with the original aspirate and biopsy. In a preferred embodiment, in vivo proliferation is assessed by reactivity to an antibody directed against a protein known to be present in higher concentrations in proliferating cells than in non-proliferating cells, such as proliferating cell nuclear antigen (PCNA, or cyclin). Such antibodies are commercially available from a number of sources, including Zymed Laboratories (South San Francisco, Calif., USA).
In a preferred embodiment, isolated MSC are placed into Dulbecco""s medium MEM (DMEM-LG) (Gibco, Grand Island, N.Y., USA). The cells are purified by a series of steps. Initially, the cells are pelleted and resuspended in Complete Medium. The cells are centrifuged through a 70% Percoll (Sigma Corporation, St. Louis, Mo., USA) gradient at 460xc3x97g for 15 minutes, the top 25% of the gradients are transferred to a tube containing 30 ml of Complete Medium and centrifuged to pellet the cells, which will then be resuspended in Complete Medium, counted and diluted to seed in 100-mm plates at 50xc3x97106 nucleated cells per plate.
In a further preferred embodiment, purified MSC are cultured in Complete Medium at 37xc2x0 C. in a humidified atmosphere containing 95% air and 5% CO2 and the cells are allowed to attach for 3 days, whereupon non-adherent cells are removed by changing the culture medium. Cellular proliferation of adherent cells and the presence of normal MSC morpholgy are assessed by phase microscopy at various time points during the subsequent growth period. Subsequent medium changes are performed every four days. When the primary cultures are nearly confluent, the cells are detached with 0.25% trypsin containing 0.1 mM EDTA (Gibco) and either diluted and replated as second passage cells, or used for reinfusion into a subject. Preferably, cells are rinsed free of culture fluid using Tyrode""s solution (Gibco). After the final rinse, cells are placed in Tyrode""s solution and placed in an incubator at 37xc2x0 C. for one hour in order to shed serum proteins. The Tyrode""s solution is removed and the cells are preferably placed into TC199 medium (Gibco) supplemented with 1% serum albumin. The cells are rinsed a number of times with this medium and after the final rinse MSC are resuspended in TC199 plus 1% serum albumin. Subsequently, MSC are injected slowly intravenously over 15 minutes. Evaluation of subsequent bone marrow aspirates are conducted up to 8 weeks after injection.
In a preferred embodiment, assessment of the in vivo proliferative effect of angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII, AII analogues, AII fragments and analogues thereof and/or AII AT2 type 2 receptor agonists on MSC and mesenchymal lineage-specific cells is done by histochemical evaluations of various tissues. In a preferred embodiment, in vivo proliferation of MSC and mesenchymal lineage-specific cells is assessed by reactivity to an antibody directed against a protein known to be present in higher concentrations in proliferating cells than in non-proliferating cells, such as proliferating cell nuclear antigen (PCNA, or cyclin; Zymed Laboratories).
In a further aspect of the present invention, the effect of angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII, AII analogues, AII fragments and analogues thereof and/or AII AT2 type 2 receptor agonists on HPC, HLSC differentiation are assessed by examination of changes in gene expression, phenotype, morphology, or any other method that distinguishes a cell undergoing differentiation from a lineage-specific cell. In a preferred embodiment, macrophage differentiation to an elicited or activated state is assessed after exposure to angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII, AII analogues, AII fragments and analogues thereof and/or AII AT2 type 2 receptor agonists, as described above. The macrophages are assessed for phagocytotic ability by any of the well known art methods, including but not limited to determination of the number of macrophages that have ingested opsonized yeast particles, and the number of yeast per macrophage ingested. (Rodgers and Xiong, Fundamental and Applied Toxicology 33:100-108 (1996)).
In another preferred embodiment, the respiratory burst activity of leukocytes is assessed after exposure to angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII, AII analogues, AII fragments and analogues thereof and/or AII AT2 type 2 receptor agonists as described above. The leukocytes are assessed for respiratory burst activity by any method known in the art including, but not limited to, the ability to generate hydrogen peroxide via the respiratory burst system. (Rodgers et al., International Journal of Immunopharmacology 10:111-120 (1988); Rodgers, In: Modern Methods in Immunotoxicology (ed. G. Burleson), Wiley Liss 2:67-77 (1995)).
Macrophage activation by the compounds of the present invention can be utilized for treating viral and microbial infections, stimulating macrophages to produce cytokines made by activated macrophages, improving macrophage presentation to T cells, and augmenting anti-tumor immunity in a mammal.
In another aspect of the present invention angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII, AII analogues, AII fragments and analogues thereof and/or AII AT2 type 2 receptor agonists are used to increase in vivo HPC, HLSC, MSC and mesenchymal lineage-specific cell proliferation. For use in increasing proliferation of HPC, HLSCP, MSC and mesenchymal lineage-specific cells, angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII, AII analogues, AII fragments and analogues thereof and/or AII AT2 type 2 receptor agonists may be administered by any suitable route, including orally, parentally, by inhalation spray, rectally, or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles. The term parenteral as used herein includes, subcutaneous, intravenous, intramuscular, intrastemal, intratendinous, intraspinal, intracranial, intrathoracic, infusion techniques or intraperitoneally.
In a further embodiment of the present invention, a method of increasing in vivo HPC, HLSC, MSC and lineage-specific mesenchymal cell proliferation by exposure to angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII, AII analogues, AII fragments and analogues thereof and/or AII AT2 type 2 receptor agonists is disclosed, either in the presence or absence of other growth factors and cytokines. Examples of such growth factors and cytokines include, but are not limited to lympholines, interleukinsxe2x80x941, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, granulocyte colony-stimulating factor, granulocyte/macrophage colony stimulating factor, macrophage colony-stimulating factor, tumor necrosis factor, epidermal growth factor, fibroblast growth factor, platelet derived growth factor, transforming growth factor beta, and stem cell factor.
The angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII, AII analogues, AII fragments and analogues thereof and/or AII AT2 type 2 receptor agonists may be made up in a solid form (including granules, powders or suppositories) or in a liquid form (e.g., solutions, suspensions, or emulsions). The compounds of the invention may be applied in a variety of solutions. Suitable solutions for use in accordance with the invention are sterile, dissolve sufficient amounts of the peptide, and are not harmful for the proposed application. In this regard, the compounds of the present invention are very stable but are hydrolyzed by strong acids and bases. The compounds of the present invention are soluble in organic solvents and in aqueous solutions at pH 5-8.
The angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII, AII analogues, AII fragments and analogues thereof and/or AII AT2 type 2 receptor agonists may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers etc.
While angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII, AII analogues, AII fragments and analogues thereof and/or AII AT2 type 2 receptor agonists can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more other agents. When administered as a combination, the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.
For administration, the angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII, AII analogues, AII fragments and analogues thereof and/or AII AT2 type 2 receptor agonists are ordinarily combined with one or more adjuvants appropriate for the indicated route of administration. The compounds may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine, and/or polyvinyl alcohol, and tableted or encapsulated for conventional administration. Alternatively, the compounds of this invention may be dissolved in saline, water, polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers. Other adjuvants and modes of administration are well known in the pharmaceutical art. The carrier or diluent may include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art.
Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin (e.g., liniments, lotions, ointments, creams, or pastes) and drops suitable for administration to the eye, ear, or nose.
The dosage regimen for increasing in vivo proliferation or differentiation of HPC, HLSCP, MSC and lineage-specific mesenchymal cell with angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII, AII analogues, AII fragments and analogues thereof and/or AII AT2 type 2 receptor agonists is based on a variety of factors, including the type of injury, the age, weight, sex, medical condition of the individual, the severity of the condition, the route of administration, and the particular compound employed. Thus, the dosage regimen may vary widely, but can be determined routinely by a physician using standard methods. Dosage levels of the order of between 0.1 ng/kg and 1 mg/kg angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII, AII analogues, AII fragments and analogues thereof and/or AII AT2 type 2 receptor agonists per body weight are useful for all methods of use disclosed herein.
The treatment regime will vary depending on the disease being treated, based on a variety of factors, including the type of injury, the age, weight, sex, medical condition of the individual, the severity of the condition, the route of administration, and the particular compound employed. For example, angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII, AII analogues, AII fragments and analogues thereof and/or AII AT2 type 2 receptor agonists are administered to an oncology patient for up to 30 days prior to a course of radiation therapy. The therapy is administered for 1 to 6 times per day at dosages as described above. In another example, in order to mobilize hematopoietic lineage-specific cells for donation by plasmapheresis the therapy is for up to 30 days for 1 to 6 times per day.
In a preferred embodiment of the present invention, the angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII, AII analogues, AII fragments and analogues thereof and/or AII AT2 type 2 receptor agonist is administered topically. A suitable topical dose of active ingredient of angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII, AII analogues, AII fragments and analogues thereof and/or AII AT2 type 2 receptor agonists is preferably between about 0.1 ng/ml and about 1 mg/ml administered twice daily. For topical administration, the active ingredient may comprise from 0.0001% to 10% w/w, e.g., from 1% to 2% by weight of the formulation, although it may comprise as much as 10% w/w, but preferably not more than 5% w/w, and more preferably from 0.1% to 1% of the formulation.
In another aspect of the present invention, an improved cell culture medium is provided for the proliferation and differentiation of hematopoietic and mesenchymal stem and lineage-specific cells, wherein the improvement comprises addition to the cell culture medium of an effective amount of angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII, AII analogues, AII fragments and analogues thereof and/or AII AT2 type 2 receptor agonists, as described above. Any cell culture media that can support the growth of hematopoietic and mesenchymal stem and lineage-specific cells can be used with the present invention. Such cell culture media include, but are not limited to Basal Media Eagle, Dulbecco""s Modified Eagle Medium, Iscove""s Modified Dulbecco""s Medium, McCoy""s Medium, Minimum Essential Medium, F-10 Nutrient Mixtures, OPTI-MEM(copyright) Reduced-Serum Medium, RPMI Medium, and Macrophage-SFM Medium or combinations thereof.
The improved cell culture medium can be supplied in either a concentrated (ie: 10xc3x97) or non-concentrated form, and may be supplied as either a liquid, a powder, or a lyophilizate. The cell culture may be either chemically defined, or may contain a serum supplement. Culture media is commercially available from many sources, such as GIBCO BRL (Gaithersburg, Md.) and Sigma (St. Louis, Mo.)
In a further aspect, the present invention provides kits for the propagation of hematopoietic and mesenchymal stem and lineage-specific cells, wherein the kits comprise an effective amount of angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII, AII analogues, AII fragments and analogues thereof and/or AII AT2 type 2 receptor agonists, and instructions for its use as a cell culture media supplement.
In a preferred embodiment, the kits further comprise cell culture growth medium. Any cell culture media that can support the growth of hematopoietic and mesenchymal stem and lineage-specific cells can be used with the present invention. Examples of such cell culture media are described above. The cell culture medium can be supplied in either a concentrated (ie: 10xc3x97) or non-concentrated form, and may be supplied as either a liquid, a powder, or a lyophilizate. The cell culture may be either chemically defined, or may contain a serum supplement.
In a further preferred embodiment, the kit further comprises a sterile container, which can comprise either a sealed container, such as a cell culture flask, a roller bottle, or a centrifuge tube, or a non-sealed container, such as a cell culture plate or microtiter plate (Nunc; Naperville, Ill.).
In another preferred embodiment, the kit further comprises an antibiotic supplement for inclusion in the reconstituted cell growth medium. Examples of appropriate antibiotic supplements include, but are not limited to actimonycin D, FUNGIZONE(copyright), kanamycin, neomycin, nystatin, penicillin, streptomycin, or combinations thereof (GIBCO).
The present invention, by providing a method for enhanced proliferation of HPC and HLSC, will greatly increase the clinical benefits of HPC transplantation. This is true both for increased xe2x80x9cself-renewalxe2x80x9d, which will provide a larger supply of HPC capable of reconstituting the entire hematopoietic system, and for proliferation with differentiation, which will provide a larger supply of lineage-specific cells, for more rapid reconstitution of mature, functioning blood cells. Similarly, methods that increase in vivo proliferation of HPC will enhance the utility of HPC transplantation therapy by rapidly increasing local concentrations of HPC (and HLSC) in the bone marrow, and thereby more rapidly producing functioning blood cells.
Similarly, methods that increase the proliferation of MSC and mesenchymal lineage-specific cells, will greatly increase the utility of MSC therapy in the repair of skeletal tissues such as bone, cartilage, tendon and ligament. More rapid proliferation of large numbers of MSC and mesenchymal lineage-specific cells will permit more efficient delivery of high densities of these cells to a defect site and more rapid in vivo amplification in the local concentration of stem and lineage-specific cells at an appropriate repair site.
The method of the present invention also increases the potential utility of HPC and HLSC as vehicles for gene therapy in hematopoietic disorders, as well as MSC and mesenchymal lineage-specific cells as vehicles for gene therapy in skeletal disorders by more efficiently providing a large number of such cells for transfection, and also by providing a more efficient means to rapidly expand transfected HPC, HLSC, MSC and mesenchymal lineage-specific cells.